JXB Advance Access originally published online on December 6, 2006
Journal of Experimental Botany 2007 58(3):569-577; doi:10.1093/jxb/erl232
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RESEARCH PAPER |
The effect of silicon on the infection by and spread of Pythium aphanidermatum in single roots of tomato and bitter gourd
Institute of Plant Nutrition, University of Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany
* To whom correspondence should be addressed. E-mail: horst{at}pflern.uni-hannover.de
Received 9 June 2006; Revised 13 October 2006 Accepted 16 October 2006
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Abstract |
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The effect of silicon (Si) supply on the infection and spread of Pythium aphanidermatum was studied in the roots of tomato [Lycopersicon esculentum (=Solanum lycopersicum), an Si excluder] and bitter gourd (Mormodica charantia, an Si intermediate accumulator). Individual roots were mounted into PVC compartmented boxes which allowed the application of Si and zoospores to defined root zones. Two days after inoculation, root growth was recorded, and P. aphanidermatum colonization of individual root sections was determined by ELISA. In tomato as well as in bitter gourd the root tip was the root section most sensitive to P. aphanidermatum infection. Application of Si did not affect severe root-growth inhibition by P. aphanidermatum in either species. However, continuous Si supply significantly inhibited the basipetal spread of the pathogen from the infected root apex in bitter gourd but not in tomato. Si application to the roots only during pretreatment or only during/after the infection of the roots failed to inhibit the spread of P. aphanidermatum. Determination and compartmentation of Si in the roots of bitter gourd revealed that apoplastic Si was not, but symplastic Si was, associated with the ability of the plant to reduce the spread of the fungus in roots. It is concluded that accumulation of Si in the root cell walls does not represent a physical barrier to the spread of P. aphanidermatum in bitter gourd and tomato roots. The maintenance of elevated symplastic Si contents is a prerequisite for Si-enhanced resistance against P. aphanidermatum.
Key words: Bitter gourd, ELISA, pathogen resistance, Pythium aphanidermatum, silicon, tomato
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Introduction |
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Root rot, associated with Pythium spp., is a major threat for many agricultural crops (Hendrix and Campbell, 1973). Pythium aphanidermatum (Edson) has a mainly tropical distribution and is pathogenic to a wide host range (Domsch et al., 1980). It infects mainly roots of seedlings or the root tips of older plants (Hendrix and Campbell, 1973) and is known to be a good root colonizer that consistently inhibits root growth (Wulff et al., 1998). After infecting the root, P. aphanidermatum can also spread through the roots of seedlings into the hypocotyl, causing stem rot and eventually post-emergence damping-off (Jones et al., 1991b).
A typical feature of P. aphanidermatum is its ability to infect plants grown in culture solution (Moulin et al., 1994) and its spread in solution is facilitated by asexually produced zoospores (Stanghellini and Rasmussen, 1994). The zoospores are attracted by root diffusates and then attach to the root mainly in the root hair zone (Jones et al., 1991a; Grosch and Schwarz, 1998; Wulff et al., 1998) where the spore adhesion and germination is then stimulated by uronic acid (Donaldson and Deacon, 1993) and Ca2+ but not by other cations (e.g. Na+, K+, Mn2+) (Donaldson and Deacon, 1992).
Many studies report a good control of Pythium ultimum and P. aphanidermatum by amending the nutrient solution of greenhouse-grown cucumber with silicon (Si) in a concentration of 1.7 mM (Chérif and Bélanger, 1992; Chérif et al., 1994b; Bélanger et al., 1995). These concentrations are higher than the plant-available Si concentrations of most soils which are known to be generally in the range 0.5–0.65 mM (Jones and Handreck, 1967). Whether Si decreases root infection by modifying root surface characteristics and/or spore germination is not known. Despite the considerable number of studies on the interaction between Si and several species of Pythium, quantitative measurements on the spread of Pythium species at a root level as affected by Si are lacking. Si deposition in cell walls may represent a physical barrier for fungal growth as originally proposed for powdery mildew by Wagner (1940). However, based on the studies mentioned above it appears more likely that Si nutrition enhances plant resistance mechanisms against Pythium spp. (Fawe et al., 2001). Heine et al. (2005) previously studied the Si nutrition of the two plant species, tomato and bitter gourd. They demonstrated that the total root Si concentration was higher in the Si-excluder tomato than in the Si-accumulator bitter gourd. This was particularly true for the cell-wall fraction, but Si concentrations in the symplastic fraction were higher in bitter gourd. Therefore, the comparison of these plant species appeared especially suitable for studies concerning the mechanisms of Si-enhanced resistance against root diseases (Heine et al., 2005). Enhanced Si accumulation in the root cell walls could represent a mechanical barrier against the spread of Pythium spp. in the root. However, if Si primarily enhances plant disease-resistance mechanisms, Si uptake into the root symplast should be more important.
The objectives of the present study were to establish an experimental protocol, which allowed the application of Si and zoospores of P. aphanidermatum to a defined apical root section of tomato and bitter gourd plants, and to develop a methodology for the quantitative assessment of the colonization and spread of the fungus along individual roots as affected by Si supply. Of special interest was the question whether root zones with high Si status represent a barrier against the spread of the fungus.
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Materials and methods |
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Cultivation of plants
Tomato [Lycopersicon esculentum (=Solanum lycopersicum)] seeds of the variety King Kong II were germinated at 25 °C on water-soaked filter paper in the dark. After 3 d, the geminated seeds were placed in a ‘sandwich system’ between two layers of foam overlain with filter paper and supported by DIN A4 PVC (polyvinyl chloride; ThyssenKrupp Schulte, Hannover, Germany) plates. The sandwich was placed in a plastic box containing 10 mM CaSO4 and 5 µM boric acid solutions and kept in a growth chamber under the following controlled conditions: photoperiod 16/8 h; temperature 30/25 °C day/night; 70% relative humidity, 150 µmol m–2 s–1 light intensity at plant canopy level.
After 1 week, tomato seedlings were transferred to a complete nutrient solution (µM): Ca(NO3)2 (800); K2SO4 (375); MgSO4 (325); KH2PO4 (100); H3BO3 (8); CuSO4 (0.2); ZnSO4 (0.2); MnSO4 (2); Na2MoO4 (0.1); NaCl (10); NH4NO3 (200); and FeEDTA (40). Depending on the experimental approach, 1.4 mM silicic acid, freshly prepared by passing potassium silicate (BDH Chemicals, Poole, UK) through an H+ cation-exchange column, was added. After another week, tap roots were cut off to induce adventitious root formation. When adventitious roots were several centimetres long plants were used for the experiment.
Bitter gourd (Mormodica charantia) seeds of a local variety from Lion Seeds, Thailand, were heat-treated at 65 °C overnight to break dormancy and subsequently germinated in limed peat (pH 6.0) in a growth chamber under the conditions mentioned above. After 12 d, roots of bitter gourd plants were carefully washed free of substrate before transfer to a nutrient solution as described above. Bitter gourd was grown for 4 d prior to experimental use.
Inoculum production and inoculation
All isolates used were tested by the Centraalbureau voor Schimmelcultures, The Netherlands (det. 273-202), and specified as P. aphanidermatum. Zoospores were produced by a modified method of Rahimian and Banihashemi (1979). One-week-old V8 agar plates with P. aphanidermatum were cut in small strips, transferred to two Petri dishes, and flooded with 20 ml of double-distilled water (DDW). The DDW was replaced after 30 min and plates were incubated at 34 °C under continuous light (100 µmol m–2 s–1 light intensity). After 4 d, the DDW was replaced again and subsequent incubaton of the plates for 16 h at 18 °C triggered zoospore release. Spores were counted using a Fuchs–Rosenthal chamber and adjusted to a concentration of 10 000 spores ml–1 with DDW. Roots were inoculated in compartmented boxes (see below) for 2 h, renewing the solution after 1 h. Except for the first experiment (see below) zoospores were applied to the 1 cm root apex only.
Influence of Si on spore germination
The influence of Si on fungal germination was studied based on a method of Bowen et al. (1992), which was modified in order to study zoospore germination. Zoospores were produced as described above and immobilized by shaking on a vortex mixer for 20 s. After centrifugation for 5 min at 420 g at 4 °C, the supernatant was discarded and nutrient solution, which was amended or not amended with 1.4 mM silicic acid and adjusted to pH 6.0 with 0.1 M sodium hydroxide, was added to the pellet. Percentage germination was recorded after 24 h. A minimum germ-tube length of at least the diameter of the cyst was required for a positive score.
Experimental set-up for spatial sensitivity experiments
The roots of intact plants were spread in plastic trays (500 mmx250 mm) and covered with nutrient solution. One single root per plant was carefully inserted in a compartmented box made from PVC (Fig. 1). Compartments were sealed with agarose (1%). This experimental approach allowed the application of specific solutions to different root zones along an individual root. When not mentioned otherwise, the compartments were filled with nutrient solution. After application of the solutions, the compartmented boxes and the remaining root system spread in the plastic tray were covered with tin foil and sealed with parafilm to keep the roots in the dark and to avoid evaporation. After 2 d (except for the first experiment) root growth of single roots was recorded and the roots were then cut into root sections as indicated. The level of Pythium colonization of individual root sections was analysed by using the double-sandwich ELISA system described below. Three different types of experiments were carried out.
- (i) Detection of the most sensitive root zone for infection: Plants grown in nutrient solution not amended with Si were used and no Si was supplied during the experiment. Zoospores were applied to the 1 cm root apex or to the 1–2 cm root zone. After 24 h, root growth was monitored, and 1 cm root sections were harvested for the determination of P. aphanidermatum colonization.
- (ii) Si application to the whole root: Plants were grown in nutrient solution containing or not containing Si. Depending on the experiment, Si treatment with Si-amended nutrient solution (1.4 mM) was continued or discontinued after the transfer to the compartments or started directly after inoculation. Zoospores were applied to the 1 cm root apex and roots were harvested after 48 h.
- (iii) Si application only to the second root section: After pre-cultivating in Si-free nutrient solution, Si supply was started together with the P. aphanidermatum inoculation (transfer to compartments). Si in the form of silicic acid (2 mM) was applied only to the 2–5 cm root section. Zoospores were applied to the 1 cm root tip, and roots were harvested after 48 h.
- (ii) Si application to the whole root: Plants were grown in nutrient solution containing or not containing Si. Depending on the experiment, Si treatment with Si-amended nutrient solution (1.4 mM) was continued or discontinued after the transfer to the compartments or started directly after inoculation. Zoospores were applied to the 1 cm root apex and roots were harvested after 48 h.
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Quantification of Pythium colonization by ELISA
The assay was carried out following a standard protocol for the ‘double antibody sandwich technique’ (Loewe Biochemica, Sauerlach, Germany). Briefly, samples were homogenized in sample buffer and 200 µl of the homogenate obtained was transferred to wells of microplates, previously coated with a Pythium-specific polyclonal antibody (IgG). After incubation at 4 °C overnight, 200 µl of a solution containing the Pythium-specific antibody coupled to an alkaline phosphatase (IgG-AP) was added and plates were then incubated for 4 h at 37 °C. The colorimetric reaction was induced by applying 200 µl of 4-nitrophenyl phosphate (1 mg ml–1). The absorbance was measured after 1 h with a photometer (BioTek, Winooski, USA). When analysing sections of non-inoculated roots an absorbance of about 0.12 representing a background value was usually measured.
The ELISA was calibrated by measuring a dilution series of P. aphanidermatum mycelia that was obtained from cultures grown on PDA plates (Fig. 2). The relationship between ELISA adsorption and mycelia mass was best described by an exponential function. The detection limit was lower than 1 µg mycelium ml–1 corresponding to 0.2 µg mycelium sample–1.
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Si determination in root segments of bitter gourd
Bitter gourd roots mounted in compartmented boxes were inoculated or not-inoculated with P. aphanidermatum as described above. Four different Si treatments were applied: continuous supply before and after the inoculation, supply only before inoculation, supply only after inoculation, and a control treatment with no Si at all.
Two days after inoculation, the 6 cm root tip was harvested and 2 cm segments were collected in centrifugation vials (Nanosep MF; PALL Life Science, New York, NY, USA), 10–15 segments per vial. A fractionated extraction of Si was performed in order to separate symplastic and cell wall-bound Si. Root segments were frozen at –18 °C to destroy cell membranes, thawed, and centrifuged at 3000 g for 20 min. The volume of the filtrate was recorded and the concentration of monomeric Si was determined by using the molybdenum blue method as described by Heine et al. (2005). This fraction was considered as symplastic fraction 1.
The pellet was resuspended in 750 µl ethanol (96%) and homogenized at a speed of 30 oscillations per second using a ball mill (MM200; Retsch, Haan, Germany). After centrifugation at 15 000 g for 20 min, the supernatant (symplastic fraction 2) was carefully removed from the pellet (cell-wall fraction) and transferred to a new vial. The washing step was repeated once and both fractions were dried at 65 °C.
Total Si was analysed after acid digestion following the colorimetric method of Novozamsky et al. (1984) as described by Heine et al. (2005).
Statistical analysis
All experiments were conducted using a complete randomized block design. The experiments were repeated two or three times if not otherwise stated. Data analysis was performed using the GLM procedure of SAS version 8.1 (SAS Institute, 2001) considering time as a block factor, and the Tukey test (Tukey, 1953) for the comparison of means.
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Results |
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The average length of germ tubes of zoospores was not negatively affected by addition of Si to the incubation medium (Table 1).
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In tomato as well as in bitter gourd, root growth was inhibited to a greater extent when zoospores were applied to the root apex (0–1 cm) as compared with the subapical root zone (1–2 cm; Table 2). Also, in both species Pythium colonization (ELISA readings) was greater for the root apex in comparison to the subapical section (Fig. 3). This indicates that the root tip is the root section most sensitive to infection by P. aphanidermatum for both plant species. Also, in both species the inoculation of the root apex (0–1 cm) resulted in basipetal spread of the fungus as shown by significantly higher ELISA readings in the subapical root sections (1–2 cm) compared with non-inoculated control plants. By contrast, when zoospores were applied to the subapical root section the apical spread of the fungus was low.
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In the next experiment, the basipetal spread of P. aphanidermatum from the inoculated root apex as affected by Si supply was studied. Again, the application of zoospores to the root apex (0–1 cm) lead to heavy infection of the 0–2 cm root apex and a complete cessation of root growth within 48 h in both plant species independent of the Si supply (Fig 4, inserts). The higher ELISA readings in P. aphanidermatum-infected roots demonstrated the successful infection and the basipetal rapid spread of P. aphanidermatum along the roots. ELISA readings declined from the inoculated root apex to more basipetal root zones (2–4, 4–6 cm). In tomato, Si treatment did not affect either the infection (ELISA reading of the apical root section) or the spread of the fungus. However, in bitter gourd, Si treatment significantly increased the ELISA reading of the apical root section (0–1 cm), whereas the basipetal spread of the fungus was inhibited by Si as indicated by significantly lower ELISA readings in the subapical root zones (2–4, 4–6 cm).
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By contrast to continuous Si supply, the effect of Si on fungal spread in the roots of bitter gourd was not observed when the Si supply was not continuous. Neither Si supply to the complete root only during the pretreatment (Fig. 5A) nor Si supply only after transfer to the compartmented boxes (Fig. 5B) influenced the P. aphanidermatum colonization in any of the root sections sampled. Again, in both experiments the highest ELISA readings were detected in the root tips (0–2 cm) followed by a gradual decrease in the basipetal direction (2–4, 4–6 cm). As might be expected from the experiment with a continuous Si supply (Fig. 4), Si also had no effect in tomato on infection and spread of P. aphanidermatum only before or after inoculation (data not shown).
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A similar result was observed when bitter gourd plants were pregrown in Si-free medium and Si was applied only to the 2–5 cm root zone when simultaneously inoculating P. aphanidermatum at the root apex (Fig. 6). ELISA readings revealed that Si supply did not influence the growth of P. aphanidermatum within or through the Si-treated root zone (2–5 cm), although for inoculated plants the Si concentration of this zone was increased from 0.80 to 1.76 mg Si g–1 dry weight (data not shown).
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This result clearly reveals that the inhibition or lack of inhibition of the basipetal spread of Pythium in bitter gourd by Si (compare Fig. 4 with Figs 5 and 6) cannot be explained solely on the basis of the total Si concentration of the subapical root sections. Therefore, for Pythium-inoculated and non-inoculated plants, the distribution of Si among the cell wall and symplast fractions was determined after continuous or discontinued Si supply.
Continued Si nutrition (+/+Si) of non-inoculated roots (Pythium –) increased total Si contents in all root zones as compared with control roots (–/– Si, Table 3). The Si taken up was equally distributed between both compartments, resulting in similar proportions of cell-wall and symplastic Si in both treatments. Si deprivation for 2 d (+/– Si) decreased the total Si concentration, in particular in the newly grown root tip [apical (0–2 cm) and subapical zones (2–4 cm)]. By the basipetal root zones (4–6 cm) the decrease was more pronounced in the symplast than in the cell wall, presumably due to rapid translocation of symplastic Si to the shoots. By contrast, total and cell-wall Si contents decreased from the apical to the basipetal zones when roots were supplied with Si for only 2 d (–/+ Si). The symplastic Si content, however, was not different among root zones for this treatment. This might reflect a fast uptake and translocation of symplastic Si resulting in an increasing proportion of symplastic Si in the subapical and basal root sections.
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Also for inoculated roots (Pythium +), Si treatment lead to significantly higher Si concentrations in all root segments sampled. For the –/– and the +/+ Si treatments of inoculated roots, in all root segments total Si concentrations were not different from the respective segments of non-inoculated roots, but the proportion of the symplastic Si tended to be higher, especially in the apical (0–2 cm) root zone. Similar to the results of non-inoculated roots, in the treatment with discontinued Si supply (+/– Si) total Si concentrations increased from the apical towards the basipetal (4–6 cm) root zone when roots were inoculated. However, the total Si concentration in the (2–4 cm) subapical root zone was higher compared with the non-inoculated root due to higher Si content of the cell-wall fraction. This difference can be explained by the cessation of growth of Pythium-inoculated roots. As root growth stops with inoculation, the Si content in the cell walls of the (+/–) Si treatment 2 d after inoculation reflects the Si status at the time of inoculation. By contrast, the newly grown roots in the non-inoculated treatment grew without Si supply and Si concentrations are consequently lower (dilution effect). Accordingly, in the treatment with Si supply for 2 d only (–/+ Si) total Si concentrations were higher in non-inoculated as compared with inoculated roots. Also, for this treatment Si contents in the symplast were higher in the non-inoculated roots, indicating an inhibited uptake of Si due to Pythium infection. However, in the treatment with continuous Si supply (+/+ Si), Si contents in the symplast were similar in inoculated and non-inoculated roots.
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Discussion |
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The results of this study clearly revealed that Si supply to Si pretreated plants inhibits the spread of P. aphanidermatum in individual roots of bitter gourd. By contrast, no effect was observed in identical experiments with tomato. This difference in the effectiveness of Si as a controlling agent for Pythium species between tomato and Cucubitaceae confirms other studies in the literature. Several studies describe the ability of Si to enhance the resistance of Cucurbitaceae against root rot caused by P. aphanidermatum and P. ultimum (Chérif and Bélanger, 1992; Chérif et al., 1994b; Bélanger et al., 1995). Comparable results with tomato are lacking, indicating that Si failed to confer resistance in this plant species.
The Pythium-specific double-sandwich ELISA proved to be a suitable tool for the quantitative detection of P. aphanidermatum in the roots of tomato and bitter gourd. A reliable detection was possible down to 1 µg fungal mycelium ml–1 corresponding to 0.2 µg mycelium sample–1. Using a similar ELISA, Yuen et al. (1998) found a good correlation between infection levels of roots of sugar beet and beans with P. ultimum. The amount of antigen was best displayed on a log10 scale, which was also found in the present study (Fig. 1).
For both species, inoculating the root apex resulted in significantly higher infection levels as compared with inoculation of the subapical (1–2 cm) section (Table 2). Also, root growth was almost totally inhibited when the root apex was inoculated, but was only reduced to 36% for tomato and 46% for bitter gourd upon inoculation of the subapical root section (Table 2). A cessation of root growth following a successful infection with P. aphanidermatum was reported for cucumber (Wulf et al., 1998) and tomato (Grosch and Schwarz, 1998). The present results indicate that zoospores of P. aphanidermatum primarily infect roots via the root apex (Table 2; Fig. 3). Working with the same pathogen, Wulf et al. (1998) and Jones et al. (1991a) showed that zoospores accumulated primarily in the root-hair zone of cucumber and tomato. Since no root hairs were formed in nutrient solution in the present experiments, the root apex and secondary root primordia could be preferential root entry-sites for the pathogen. The latter is indicated by the observation that infection of the subapical root section lead to significantly enhanced ELISA readings in bitter gourd (Fig. 2) which showed more frequent secondary root primordia in this root zone than tomato. This confirms earlier findings that fully differentiated root tissue is less susceptible to infection by P. aphanidermatum (Martin and Loper, 1999).
Once established in the roots, the spread of Pythium spp. is affected by several factors, among them temperature (Grosch and Schwarz, 1998) and the nutritional status of the host (Hendrix and Campbell, 1973). In the case of bitter gourd, Si supply proved to be an important factor influencing the spread of P. aphanidermatum. After infection of the root apex, infection levels were significantly lower compared with –Si plants in the subapical root sections 2–4 cm and 4–6 cm when plants were continuously supplied with Si (Fig. 3). Surprisingly, in the root tip, infection levels were found to be higher in bitter gourd supplied with Si. There could be two reasons for this: (i) the growth of the fungus was restricted to this section due to the inhibition of spreading to and colonizing basipetal sections—the consequence would be an increased growth in the root tip where the infection threat was so high that Si was not effective in suppressing fungal growth; (ii) Si enhanced zoospore germination leading to a higher number of penetration pegs owing to a stimulation of zoospores in the presence of Si. Information on the effects of mineral nutrients on spore germination is rare. Bowen et al. (1992) observed a stimulation of Si treatment on in vitro conidial germination of Uncimala necata, whereas Menzies et al. (1992) did not find an influence of Si on the germination of Sphaerotheca fuligena. Zoospore germination of P. aphanidermatum was stimulated by high concentrations of Ca2+, Mg2+, and Sr2+ (Donaldson and Deacon, 1992). In the current study, no influence of Si on zoospore germination of P. aphanidermatum was found (Table 1).
It was proposed for leaves that Si deposition in the cell walls constitute a mechanical barrier which impedes the penetration of fungal hyphae (Heath, 1979; Heath and Stumpf, 1986). The present results do not support this hypothesis for the infection of bitter gourd roots by P. aphanidermatum: no influence of Si on the fungal spread was observed when Si was applied only at the time of infection (Figs 5B, 6) even though Si concentrations were greatly increased in the Si treatment (Table 3). The same conclusion can be drawn from the experiments in which plants were pretreated with Si, but Si application was discontinued during inoculation (Fig. 5A). Even though Si was present in the roots of pretreated plants, Si failed to decrease fungal spread. Kauss et al. (2003) identified a gene encoding a protein that polymerizes silicic acid to insoluble silica at the infection site of Colletotrichum lagenarium in the leaves of cucumber, thus creating a barrier against fungal spread. However, in the roots of bitter gourd, an infection with P. aphanidermatum did not increase the Si content (Table 3) making a similar mechanism unlikely.
The mechanical barrier theory with regard to Si in roots was ruled out as a disease mechanism of cucumber against P. ultimum (Chérif et al., 1992a, b). These authors emphasized a metabolic role of Si in disease resistance of cucumber by stimulating plant natural defence mechanisms. They observed a Si-enhanced activation of peroxidases and polyphenoloxidases (Chérif et al., 1994a) and accumulation of fungitoxic phenolic compounds (Chérif et al., 1992a) after infection with P. ultimum. They concluded that Si induced plant-host resistance (Fawe et al., 2001). This is also suggested by the present results on the P. aphanidermatum/bitter gourd pathosystem where Si treatment restricted the basipetal spread of the fungus from the infected root apex (Fig. 4B). As in the case of well-known activators of systemically acquired resistance like DL-3-aminobutyric acid (Juen et al., 2000) or potassium phosphate (Mucharromah and Kuc, 1991), Si-induced defence mechanisms are activated after a lag phase; no Si effect was observed when Si supply started immediately after inoculation with P. aphanidermatum (Fig. 5B). However, the effect of Si was rapidly lost after Si was withdrawn from the nutrient solution (Fig. 5A). Kauss et al. (1993) demonstrated that the efficacy of an elicitor of systemically acquired resistance depends on its resistance to degradation. The present results suggest that the observed Si-enhanced resistance of bitter gourd against P. aphanidermatum is linked to the maintenance of a high Si status in the root symplast. Only under a continuous Si supply before and after inoculation did Si concentrations in the symplast remain high (Table 3). A rapid decrease of the symplastic Si concentration in the apical and subapical root sections after discontinuing the Si supply could be explained by a rapid translocation to the shoot (Heine et al., 2005). The application of Si after P. aphanidermatum root infection failed to increase the symplastic Si concentration probably due to inhibition of Si uptake by the heavily damaged root apices.
A rapid decline of Si-induced resistance after transfer of the plants to Si-free solution was also reported for the pathosystem S. fuligena/cucumber (Samuels et al., 1991). These authors postulated that the soluble silicic acid in the leaf tissue stimulates the defence mechanism, whereas residual polymerized Si had no effect on the pathogen. For the Si-enhanced resistance of cucumber against P. ultimum it was concluded that protection was not related to the total Si concentration in the root tissue but rather to the availability of mobile silicic acid at the time of infection (Chérif and Bélanger, 1992).
Si did not affect the spread of P. aphanidermatum in the roots of tomato, regardless of the experimental approach used (Fig. 4A). Previous experiments had shown that the application of Si increases the Si concentrations in roots of tomato more than in bitter gourd (Heine et al., 2005). The difference between the plant species in the role of Si in resistance against P. aphanidermatum further supports the idea that the beneficial effect of Si on plant health is not primarily dependent on bulk-tissue Si concentration. The ability of Si to induce resistance appears to be rather linked to its cellular compartmentation within the root tissue. Using a fractionating method for root Si, Heine et al. (2005) demonstrated that tomato is characterized by Si accumulation in the cell-wall fraction of roots. However, fungal spread in the roots did not differ between –Si- and +Si-treated plants. On the other hand, fungal spread was significantly inhibited in bitter gourd which maintained a higher symplastic Si concentration in spite of lower total bulk-tissue Si concentrations in the roots.
From the results presented it is concluded that the ability of Si to inhibit fungal spread in root apices depends on the uptake of Si into the root symplast. Si accumulation in the cell walls of bitter gourd seems to have no influence on disease progress. However, the physiological and molecular basis of Si-mediated resistance is still not well understood (Fauteux et al., 2005).
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